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The juvenile hormones: historical facts and speculations on future

research directions

Lawrence I. Gilbert

a,*

_{, Noelle A. Granger}

b

_{, R. Michael Roe}

c

a_{Department of Biology, Campus Box #3280 Coker Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA} b_{Department of Cell Biology and Anatomy, Campus Box #7090, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090,}

USA

c_{Department of Entomology, Campus Box 7647, North Carolina State University, Raleigh, NC 27695-7647, USA}

Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000

1. Historical aspects

In all of endocrinology there is no more wondrous name for a hormone than the insect juvenile hormone (JH). Could V.B. Wigglesworth have predicted some six decades ago that his term “juvenile hormone” would offer promise of immortal youth to the aged, the expec-tation of a bloom of dollars to agrochemical concerns, and the hope of solutions to basic problems by develop-mental biologists and entomologists. The aged have been disappointed and the high expectations of commercial firms have not been met, but hope remains that JH can be used as a probe to ultimately solve basic questions in development.

It has been more than two centuries since Lyonet (1762) described granulated vessels in the thorax of lepi-dopteran larvae that proved to be the prothoracic glands. By contrast, the corpora allata were not mentioned in the literature until Mu¨ller (1828) described organs in the cockroach that he called pharyngeal bodies and which he thought innervated the dorsal vessel and esophagus. During the remainder of the 19th century, the corpora allata were described as sympathetic ganglia or other components of the nervous system, as indicated by the various descriptive terms given them, e.g. accessory gan-glia, tracheal gangan-glia, lateral ganglion, lateral head gang-lion, appendage of the pharyngeal ganggang-lion, etc. In 1899, Heymons dubbed these organs the corpora allata and correctly described their embryological origin, but also believed that they were a pair of sympathetic ganglia concerned with the innervation of the digestive system.

* Corresponding author. Tel.:+1-919-966-2055; fax:+ 1-919-962-1344.

E-mail address: lgilbert@unc.edu (L.I. Gilbert).

0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 3 4 - 5

In 1910, Police suggested that the corpora allata were endocrine organs in phasmids, but that they probably had a nervous function in other insects.

It was Nabert in 1913 who, on the basis of studying a variety of insects representing several orders, finally stated that the corpora allata were glandular and exhib-ited periodic internal secretions. This was confirmed by the work of Ito (1918), who concluded that the corpora allata were indeed organs of internal secretion and that they functioned in the adult moth as well. All of this work was anatomical and histological with a bit of microsurgery, with the corpora allata being observed under the light microscope using a variety of stains. Ito was probably the first to use planimetrics to measure the diameter of the corpora allata and to demonstrate variations in size during various stages of metamor-phosis.

Burtt (1937, 1938) described the corpora allata in the higher Diptera and found that these organs were a por-tion of the “Weissman’s Ring” or as it is now known, the ring gland (Burtt and Hadorn, 1937). He hypothes-ized that certain structures within the ring gland rep-resented modified and fused corpora allata. We now know, of course, that the ring gland is composed of cells with the function of the corpora allata, cells with the function of the prothoracic gland, and an anatomical por-tion that appears to have elements of the corpora car-diaca (e.g. Dai and Gilbert, 1991). It was about that time that V.B. Wigglesworth (1935) began his historic studies on many aspects of Rhodnius development and meta-morphosis, making efficient use of surgical techniques such as decapitation and subsequent parabiotic regimens. He established the critical period for molting in this bug, and because the corpus allatum displayed cyclical activity which was correlated with this critical period, he assumed at first that this gland was the source of the

molting hormone. Further surgical studies suggested that the insect also contained an “inhibitory factor” which prevented the first four larval stages from molting directly into adults. In 1935 he suggested that both the molting and inhibitory hormones were produced by the corpus allatum, and in 1936 his classic paper on the function of the corpus allatum in the growth of Rhodnius was published. He showed that the corpus allatum was the source of the inhibitory hormone that prevents meta-morphosis in young larvae and that corpora allata from these young larvae when implanted into fifth instars caused them to undergo a supernumerary molt. He per-fected the surgery of allatectomy and showed that such animals could molt, indicating that the corpus allatum was not the source of molting hormone. Wigglesworth concluded that each cell has the potential for larval or adult differentiation and that hormone titers determine which potential is realized. It is the concentration of the corpus allatum hormone that determines the extent of metamorphosis at the next molt, wrote Wigglesworth (1948), and of course, analogous experiments were done on other insects over the next twenty-five years showing that Professor Wigglesworth was correct. It is of interest that some of his work on this subject probably would not be accepted for publication today because of the small sample size used, i.e. only two of the five operated fifth instars molted into intermediates after receiving cor-pus allatum implants from fourth instars. Our own per-sonal bias is that Wigglesworth had the best intuition of any scientist we have met or read about.

In the first of many studies of JH function, Piepho (1938a,b, 1950) showed via histological studies that the formation and nature of the cuticle of Galleria was under hormonal control. In the initial experiments, the integu-ment from one larva was grafted to another, and the graft integument molted with the host. This led to a series of experiments in which a fragment of integument from one larva was implanted into the abdominal cavity of another. The epidermis of the fragment regenerated around the cuticle of the implant to give rise to an epi-dermal vesicle with cuticle at its interior. Piepho’s lab-oratory showed that when the host molted, the vesicle molted in concert. His studies very beautifully demon-strated that molting was under hormonal control, that the epidermis reacts to JH by laying down larval structures (cuticle) and that the epidermis does not “count the molts”. The classic study of Piepho (1942, 1950) revealed important phenomena concerning tissue com-petence and tissue responsiveness. First, it appeared that the epidermal cells remained responsive to JH until very late in the last stadium. When implants were done very late in the stadium, there was a patch of larval cuticle at the wound site in the resulting pupa. Second, the results showed that wounded epidermis is much more sensitive to the action of the JH than intact regions. This was a vital finding for the development of the Galleria wax

test that was used successfully for some twenty years before analytical analyses of JH came to the fore (Gilbert and Schneiderman, 1960; Schneiderman and Gilbert, 1964), and allowed the first semiquantitative titer of JH (Gilbert and Schneiderman, 1961b; Table 1).

During that same era, the classic studies of Bounhiol (1936–1938) showed the effects of corpora allata extir-pation in Lepidoptera (Bombyx). Bounhiol’s results were confirmed and extended by those of Fukuda (1942); Fukuda (1944). It is of interest that despite the use of HPLC, mass spectrometry, etc., to measure JH, our con-cept of how the changing titer affects developmental pro-cesses has not advanced a great deal over the past fifty years.

The “modern” era of JH research began with the criti-cal finding by Carroll Williams (1956) that the male

Hyalophora cecropia moth contained a store of a

lip-oidal “golden oil” with JH activity when assayed on lepi-dopteran pupae. The original intent of the experiment was to determine if the life of the male saturniid moth could be extended if it were parabiosed to a pupa. As is so common in biology, serendipity ruled, and the pupa molted into a second pupa, indicating that the moth had furnished the pupa with JH. Furthermore, Williams’ dis-covery elicited a frenzy of studies of JH in a variety of insects — for the first time, an active extract of this amazing hormone was available and it worked on most insect species. Indeed, from one male H. cecropia abdo-men enough JH could be extracted and diluted in peanut oil or mineral oil to conduct hundreds or even thousands of experiments. If you could find one male moth, you were in business for life! If Williams had used the other saturniid studied in his laboratory in his original experi-ments, the parabiotic pupal partner would have molted into an adult since the adult Antheraea polyphemus is almost devoid of JH. In retrospect, it is clear from these early experiments that Williams, Wigglesworth, Piepho, etc. all had an almost uncanny ability to interpret cor-rectly from unexpected, incomplete and seemingly bizarre results.

It was the patience and persistence of a young German scientist, Herbert Ro¨ller, that allowed his group to finally deduce the structure of JH I (Ro¨ller et al., 1967). At the time, there was competition between the Ro¨ller labora-tory and that of Howard Schneiderman, the latter con-firming the structure of JH I and identifying JH II several years later (Meyer et al., 1970). The difference between the approach of the two laboratories was that Ro¨ller et al. used glass columns in their gas chromatographic analyses while the Schneiderman group used all metal columns, which enhanced the lability of the JH mol-ecule. In any event, pure JHs were available about thirty years ago.

It should be noted that we have chosen only three top-ics for our review and speculations. These are: control of the corpora allata; control of JH titer; and JH action

Table 1

Juvenile hormone content during the life history of the Cecropia silkworma

Stage Approximate juvenile Approximate juvenile Approximate juvenile

hormone concentration of hormone concentraion per hormone activity per gram extract: (Cecropia insect or fragment: fresh weight: (Cecropia units/gram extract) (Cecropia units/animal or units/gram animal or

fragment) fragment fresh weight)

Unfertilized eggs Minimum of: 165 0.042 8.151

Unfertilized eggs from alatectomized “0” “0” “0”

7-day embryos Minimum of: 165 0.031 7.095

7-day embryos from allatectomized Minimum of: 70 0.012 3.108

1st instar larvae (newly hatched) Minimum of: 165 0.031 7.475

5th instar larvae (mixed sex) 35 4.162 0.571

Diapausing pupae (1 month old) 20 5.780 1.468

Diapausing pupae (1 month old) 20 6.300 1.114

Chilled pupae (6 months old) “0” “0” “0”

Chilled pupae (6 months old) “0” “0” “0”

2-day old developing adults “0” “0” “0”

8-day old developing adults “0” “0” “0”

11-day old developing adults “0” “0” “0”

14-day old developing adults “0” “0” “0”

17-day old developing adults “0” “0” “0”

20-day old developing adults 125 25 13.70

22-day old developing adults 400 122.68 201.64

Adult (4 days) 675 151.88 298.75

Adult (7 days) 1000 178.30 418.00

Adult (7 days) 125 17.50 11.63

a_{Activity of extracts is expressed in Cecropia units. One Cecropia unit is equivalent to the juvenile hormone activity found in one milligram}

of extract obtained from the abdomens of seven-day old male Cecropia moths. Extractions of developing adults and adult moths were conducted on the abdomens only and the activity noted is for the abdominal extract. From Gilbert and Schneiderman (1961b).

(receptors). We simply could not summarize or do jus-tice to all the presentations made during the symposium and chose topics we felt were important and with which we were personally involved. We apologize to those individuals we did not cite since it is possible that their contributions may be even more important than those summarized here.

2. Control of the corpora allata

Changes in the JH titer, which regulate the growth and development of immature insects and reproduction in adults, are controlled precisely by various physiological and biochemical processes i.e. synthesis, degradation, sequestration and secretion. Of these, the regulation of synthesis has generally been considered the most important, and a large body of evidence for both stimu-latory (allatotropic) and inhibitory (allatostatic) control of JH synthesis by the corpus allatum has accumulated from many years of studies in vivo.

2.1. Allatotropins (ATs.)

Since the corpora allata are innervated by the axons of both peptidergic neurosecretory cells and typical neurons located within the brain, the mechanisms stimulating and

inhibiting corpora allata function promised to be com-plex from the outset. Research in this area in the last decade has confirmed this prediction. The primary focus of this work has been on regulatory peptides, termed allatotropins and allatostatins. With regard to allatotrop-ins, despite a wealth of indirect evidence for the stimu-lation of JH production by neural factors, particularly during larval development, only one had been isolated and sequenced prior to this conference, an allatotropin from adult Manduca (Mas-AT) (Kataoka et al., 1989). This amidated tridecapeptide stimulates JH biosynthesis only by the corpora allata of adult Manduca. The gene

Mas-AT is expressed as three different mRNAs which

differ from one another by alternative splicing and which have been suggested to encode three distinct prohor-mones (Taylor et al., 1996). Immunocytochemistry using polyclonal antibodies to Mas-AT and in situ hybridiz-ation with riboprobes for its mRNAs have been used to demonstrate that Mas-AT does exist in larvae, with its greatest abundance in two cells in the frontal ganglion that project to the gut and in cells in the terminal abdominal ganglion (Bhatt and Horodyski, 1999). Only low levels were found in the brain (in non-neurosecre-tory cells) and subesophageal ganglion. This result con-trasts with that of Zˇ itnˇan et al. (1995), who used anti-bodies to the peptide to demonstrate Mas-AT in a limited number of cerebral neurons in the larva, plus axons in

the corpora cardiaca and corpora allata. Thus, although Mas-AT has no apparent effect on the larval corpora allata of Manduca, it must have some other unknown function at this stage. Interestingly, while Mas-AT does stimulate adult corpora allata, its mRNAs were not detected in the prepupal, pharate adult or adult brain, but were present in the pterothoracic and unfused abdominal ganglia of the pupa (Bhatt and Horodyski, 1999). A recent study by Lee et al. (1998) demonstrated the inhi-bition of midgut ion transport in Manduca fifth instars by Mass-AT, an effect which appears to be species-spe-cific. It has another effect as well, exhibiting cardioacce-leratory activity in the pharate adult, but not the larva (Veenstra et al., 1994). These observations are illustra-tive of a phenomenon observed with a number of other insect neuropeptides i.e. different effects which are both stage- and tissue-specific. As was discovered with allato-statins, these effects can be species-specific as well (see below).

In the decade since the identification of Mas-AT, no allatotropin sequences were published for any other species, and until recently, there was a dearth of work on other putative allatotropins. The exceptions are the demonstration of allatotropic activity in the subeso-phageal ganglion of crickets (Lorenz and Hoffmann, 1995), and some recent work with Galleria (Bogus and Scheller, 1996). In the latter study, monoclonal anti-bodies raised to a protein fraction of larval brains that stimulated JH II synthesis by corpora allata in vivo and in vitro, were used to identify a single 20 kDa peptide by immunoblotting. Immunoreactivity was also observed in two pairs of cerebral neurosecretory cells and in cells of the corpus cardiacum. Although the size of this puta-tive allatotropin makes it an unlikely homolog of Mas-AT, a pair of immunoreactive median cerebral neuro-secretory cells were observed with antibodies to Mas-AT. This leads to the suggestion that Mas-AT and

Gal-leria AT have common epitopes due to splicing from

the same preprohormone.

A significant outcome of this conference is direct evi-dence from several different studies showing that Mas-AT is a functional moiety in other Lepidoptera (see Stay, this symposium). A stimulatory peptide in methanol extracts of adult brains of Spodoptera frugiperida, the fall army worm, has been identified by Edman degra-dation and mass spectrometry as Mas-AT (Oeh et al., this symposium). Exposure of adult corpora allata to synthetic Mas-AT results in a strong, dose-dependent and reversible stimulation of JH biosynthesis. The most interesting observation of this study is that a synthetic

Manduca allatostatin (Mas-AS) did not affect the rate of

JH production by Spodoptera glands but inhibited

Mas-AT-stimulated synthesis. In the tomato moth, Lacanobia oleracea, larval corpora allata can be stimulated and

inhibited, respectively, by synthetic AT and Mas-AS, in contrast to Manduca, where only Mas-AS is

effective (Audsley et al., this symposium). Furthermore, a Mas-AT-like peptide has been identified by ELISA in extracts of larval brains of this species. Genes containing sequences identical to those for Mas-AT and Mas-AS have been cloned in Pseudaletia unipunctata, the true armyworm moth (Truesdell et al., this symposium), and immunocytochemical analysis with antibodies to Mas-AT have revealed immunoreactivity in the brain, abdominal ganglia, and corpora allata of Pseudaletia adults. Finally, Mas-AT stimulates the corpora allata of honey bee larvae in a dose-dependent, reversible, and stage-specific manner, suggesting that this peptide could be a regulatory factor in Hymenoptera as well (Rachinsky et al., this symposium).

2.2. Non-neural allatotropins

If Mas-AT does not stimulate larval glands in

Mand-uca, what does? Perhaps, as seen in Spodoptera, the

activity of one peptide is only detected in the presence of the other. Thus in Manduca, Mas-AT may only stimulate allatostatin-inhibited glands. Alternatively, stimulation may occur as a result of the absence of the allatostatin or the presence of an AT-like factor from another tissue source. It has been suggested for Diploptera that the absence of allatostatins results in the stimulation of JH biosynthesis, and that certainly could suggest the pres-ence of an allatotopin in certain experiments in vivo. Alternatively, the source of a stimulatory factor could be other than neural tissues (see Stay, this symposium). The ovaries have been indicated as a source of allatotro-pin in Diploptera (see Stay, this symposium), in Grillus

bimaculatus (Hoffmann et al., 1996), and the male

accessory sex gland in Drosophila (Moshitsky et al., 1996). As reported at this conference, the sex peptide of the medfly Ceratitis capitata stimulates the corpora allata to produce a lipoidal molecule related to JH (Moshitsky et al., this symposium). In Diploptera, the ovarian factor appears to be proteinaceous and water sol-uble, with a molecular weight of less than 10 kDa (Unnithan et al., 1998). Ovary-preconditioned medium stimulates corpora allata activity in vitro, but only when the glands’ neural connections to the brain are severed, further complicating the picture of gland control in this species.

2.3. Future research on allatotropins

In summary, there are sufficient gaps in our knowl-edge of insect allatotropins to indicate that there is both the opportunity and the need for more work in this area. It is clear from the most recent studies that Mas-AT may be ubiquitous in Lepidoptera, much as the dipteran alla-tostatin (Dip-AS) is in different insect orders. The mol-ecular basis for these similarities needs to be established. The fact that Mas-AT has functions other than the

stimu-lation of JH production mirrors the trend observed for the allatostatins (see below) and also deserves further investigation. Is the allatotropic effect the primary func-tion of the molecule? Perhaps most importantly, the interactions between allatostatins and allatotropins in the control of the corpora allata are fertile, but unexplored, territory, and should lead to studies of their receptors and the downstream events controlled via the ligand-receptor interaction. It is these paths that typify the most recent work on allatostatins.

2.4. Allatostatins (AS)

In contrast to the slowly developing field of research on allatotropins, there has been an explosion of research on allatostatins, which inhibit JH synthesis, beginning with the seminal work of Stay and Tobe on the allatostat-ins of Diploptera punctata (Stay et al., 1991). At the time of the Sixth Conference on the Juvenile Hormones, 13 allatostatins had been identified in Diploptera, each with a similar C terminus: Y/F-X-F-G-L/I-NH2, with one

terminating in isoleucine. All of the Diploptera allatosta-tins appear to be produced in the same cell, and all inhibit JH synthesis in vitro but with different potencies, ranging in concentration from an ED50 of 102

11 _{M to}

1027 _{M (Stay et al., 1996). Localization of allatostatin}

immunoreactivity in the nervous system of Diploptera revealed numerous immunoreactive neurons in the brain and also in all other ganglia (Stay et al., 1994; Stay, this symposium). The arborization of the axons of cerebral allatostatin immunoreactive cells within the corpora car-diaca suggested that allatostatins could be released from this neurohemal organ, and allatostatin I was found in the hemolymph. Thus the corpora allata of Diploptera appear to be regulated by allatostatins delivered directly to the gland via the axons of the lateral neurosecretory cells of the brain or indirectly via the hemolymph.

A Manduca allatostatin (Mas-AS) was purified and sequenced from pharate adult heads in 1991 (Kramer et al., 1991). Its sequence bears no homology to the YXFGLamide family of allatostatins, but it inhibits JH biosynthesis reversibly in both adults and larvae, with an ED50value of 2–5 nM with corpora allata from day

0 fifth instars. An immunohistochemical demonstration of Mas-AS revealed two groups of lateral neurosecretory cells in brains of last instars, and arborization of the axons of one group (Ib) within the corpora allata (Zˇ itnˇan et al., 1995).

Another factor has been identified in Manduca which also inhibits the corpora allata, but which does so in a non-reversible fashion (Bhaskaran et al., 1990). This cer-ebral factor, termed an allatinhibin, provides stable inhi-bition when corpora allata are treated with the factor in vitro and then implanted into penultimate instar larvae (Unni et al., 1993). Its structure is unknown.

2.4.1. Allatostatins in other species

At the time of the last JH conference, 30 different peptides belonging to the allatostatin family had been identified in several different insect species. Since that time a considerable number have been added to the list (see Stay, this symposium), and members of the allatos-tatin family have been identified by immunoreactivity in Crustacea as well: crab, lobster, and crayfish (Skiebe, 1999). Allatostatins with the typical C terminus sequence and an inhibitory effect on JH synthesis have been ident-ified in two other cockroaches, Periplaneta americana (Weaver et al., 1994) and Blatella germanica (Belles et al., 1994) and two crickets, Gryllus bimaculatus and

Acheta domestica (Lorenz and Hoffmann, 1995). Three

structurally similar allatostatins for which a function(s) has not yet been determined, also have been isolated from the mosquito, Aedes aegypti (Veenstra et al., 1997). In addition, structurally identical peptides were isolated from the blowfly, Calliphora vomitoria, where they inhibited corpora allata activity (Duve and Thorpe, 1994; Duve et al., 1996) and from the honeybee, Apis

melli-fera, where they did not (Rachinsky and Feldlaufer,

2000). As reported at this conference, multiple allatos-tatic peptides have been isolated from the brains of the stick insect (Lorenz et al., this symposium) and are identical to those in Diploptera and Schistocerca. How-ever, while these peptides strongly inhibit JH synthesis in crickets, they have no effect on the corpora allata of the stick insect, suggesting a primary function other than the inhibition of JH biosynthesis.

2.5. Structural allatostatins with other activities

The fact that some of the allatostatins, as defined by their structure, did not function as their nomenclature implied has led to the discovery of a wide range of activities for these neuropeptides. Allatostatin-like pep-tides have frequently been found to affect muscle con-tractility, particularly in the midgut. In Diploptera, where Dip-AS immunoreactivity and mRNA synthesis have been found in endocrine cells of the midgut as well (Yu et al., 1995), allatostatins act as potent inhibitors of myogenic and proctolin-induced contractions of the midgut (Stay et al., 1994). Allatostatins also inhibit gut motility in moths (Duve et al., 1997), in addition to cock-roaches and blowflies. Recently, a new member of the YXFGLamide, or allatostatin, family, has been isolated and sequenced from the ventral nerve cord of Manduca (Davis et al., 1997). An antibody to cockroach allatosta-tin that recognizes the Manduca peptide was used to map allatostatin immunoreactive neurons in the larval ner-vous system and revealed neuroendocrine cells in the brain, abdominal ganglia and their respective neu-rohemal organs. By contrast, immunoreactive inner-vations of the corpora allata were sparse, suggesting that this peptide does not regulate corpora allata

activity. However, many thoracic motor neurons were immunoreactive, indicating a myotropic or anti-myotropic function in the larva. Interestingly, immuno-reactivity disappeared during metamorphosis and did not reappear in the adult.

Ten peptides belonging to the YXFGLamide allatosta-tin family have been identified and sequenced in

Schisto-cerca (Schoofs et al., 1997). These allatostatins, termed

schistostatins, inhibit peristaltic movements of the ovi-ducts. In the earwig, where Dip-AS 7 immunoreactivity is found predominantly in the recurrent and esophageal nerves, the last abdominal ganglion and the proctodeal nerve, Dip-AS significantly and reversibly decreased hindgut motility but had no effect on JH biosynthesis (Rankin et al., 1998a). HPLC fractions of earwig brain extract were found to contain Dip AS-like material by radioimmunoassay using antibodies to the cockroach allatostatin, and these fractions also inhibited JH biosynthesis by cockroach corpora allata and earwig hindgut motility (Rankin et al., 1998b). The demon-strated presence of allatostatins in Diploptera hemocytes (Skinner et al., 1997) suggests that they may also play an unknown role(s) in hemolymph functions. In Blatella

germanica, allatostatins inhibit vitellogenin release by

the fat body, presumably by inhibiting the process of vitellogenin glycosylation (Martin et al., 1996).

The existence of Mas-AS in other insects has also been suggested by the work of Jansons et al. (1996) and by results presented at this meeting (W.G. Bendena, M. Cusson., P. Koladich, M.D. Price, I.S. Jansons, P. Truesdell, and S.S. Tobe, unpublished observations). Brain cDNAs have been found in Pseudalecia and

Dro-sophila which specify a neuropeptide that upon

cleav-age, yields a peptide that is similar to Mas-AS. Although numerous cells in the brains of these two species are immunoreactive to antibodies to Mas-AS, synthetic Mas-AS does not affect JH biosynthesis in vitro by Drosophila ring glands and has limited ability to inhibit Pseudalecia glands in vitro (Jansons et al., 1996). The Drosophila peptide has no affect on JH biosynthesis in vitro, when tested with ring glands from third instars and day 2 adults (W.G. Bendena, personal communication). Thus in both Drosophila and

Pseuda-lecia, this peptide probably has a functional role

differ-ent from that in Manduca. Nevertheless, there is evi-dence for a cerebral allatostatin in Drosophila, based on the work of Richard et al. (1990) and Altaratz et al. (1991). As previously noted, the larval corpora allata of Lacanobia can be inhibited effectively by synthetic Mas-AS, and Mas-AS-like immunoreactivity has been detected in the central nervous system, midgut and Mal-pighian tubules of this species (Audsley et al., 1998 Audsley et al., 1999, this symposium). Its presence in sites other than the central nervous system suggests that other roles may exist for this peptide.

2.6. Control of allatostatin titer

If allatostatins can exert their effects via the hemo-lymph, how are their hemolymph titers controlled? While research on the synthesis of allatostatins and its control has not yet begun, some recent research explores their degradation. It is now known that allatostatin con-centrations are affected by a susceptibility to degradation (Bendena et al., 1997). Incubation of As 7 or Dip-AS 9 with hemolymph for 30 min revealed two primary catabolic cleavage steps: cleavage by a putative endopeptidase, yielding a C terminal hexapeptide, and subsequent cleavage of this product by an amastatin-sensitive aminopeptidase, to yield the C-terminal penta-peptide. Nevertheless, these hemolymph enzymes do not inactivate allatostatins since the C-terminal pentapeptide core (the minimal sequence necessary for the inhibition of JH synthesis) remains (Garside et al., 1997a). On the other hand, membrane preparations of brain, gut and corpora allata cleave allatostatins at the C-terminus, also in a two-step process, completely inactivating the pep-tide (Garside et al., 1997b). On the basis of information provided by the prior two studies, Nachman et al. (1999) have successfully synthesized pseudopeptide mimetic analogs of Dip-AS resistant to degradation by hemo-lymph and tissue-bound peptidases, by increasing steric hindrance of the degradative enzymes. As reported at this meeting, these analogs show varying resistance to catabolism, and all inhibit JH biosynthesis in vitro (Nachman et al., 1999; Garside et al., this symposium). Injection of mated Diploptera females with some of the analogs inhibited JH biosynthesis significantly by sub-sequently explanted corpora allata, as well as basal oocyte growth in vivo. This approach should allow more critical studies of the physiological processes modulated by allatostatins, since allatostatic activity was retained in the analogs.

2.7. Allatostatin receptors

Recent work on allatostatins has focused on their receptors, since the kind and number of receptors present in the corpora allata or any other tissue with a specific response to an allatostatin would control the timing, dur-ation, and strength of the response. Early work by Cus-son et al. (1991a) demonstrated by photoaffinity labeling the presence of two different putative receptors in

Diploptera glands. A later study, which employed an in

vitro binding assay in addition to the photoaffinity assay, identified a 37 kDa receptor for both AS-5 and Dip-AS-7 in adult female brains (Yu et al., 1995). This recep-tor had a single Kd of 9 × 10210 M for Dip-AS-5, but

two for Dip-As-7 (1.5 ×1029 _{and 3.8} ×₁₀29 _M),

sug-gesting that two receptor sites exist for Dip-AS-7. The affinity of the brain binding site for Dip-AS-5 was higher than that for Dip-AS-7, corresponding to the order of

their potencies in vitro. In the corpora allata, a single Kd

was obtained for an AS-7 receptor (7.2 × 10210_{M). A}

subsequent structure-activity study, using synthetic anal-ogs of Dip-ASB2 (Dip-AS-2), again revealed two recep-tor types in the corpora allata, based on the biphasic inhibitory response of the corpora allata to some of the ASB2 analogs (Pratt et al., 1997). These authors propose that the C-terminal portion of allatostatins contains, in addition to the “message” portion of the neuropeptide (that part responsible for a full effect), a significant amount of “address” information (binding affinity for the receptor). They conclude that a pentapeptide is likely to be the smallest fully active allatostatin structure. Diver-gent evolution of receptor types is proposed to occur together with the evolution of multiple allatostatins from a common ancestor (see below). The recognition of shorter allatostatic peptides by one receptor, and of larger peptides by the other, would allow for cross-talk between the two types of receptors, for the congruence of the “message” of all allatostatins, and for a divergence of function. Cloning and sequencing of allatostatin receptors is clearly the next step in sorting out the basis of the ubiquitous occurrence but varied functions of the multiple allatostatins.

2.8. Effect of allatostatins on the JH pathway

Where in the pathway of JH biosynthesis does the cockroach allatostatin act? Early work by Pratt et al. (1991) demonstrated that the addition of exogenous far-nesol or mevalonate reversed the allatostatic inhibition of JH biosynthesis in vitro, suggesting that the target of the allatostatin existed prior to mevalonate kinase activity. Recent work has shown that the allatostatin is a more effective inhibitor of JH biosynthesis in glands that utilize glucose or amino acids as their carbon source, rather than acetate (Sutherland and Feyereisen, 1996). These results demonstrate further that either the transport of citrate across the mitochondrial membrane (the citrate/malate shuttle) and/or the cleavage of citrate to yield cytoplasmic acetyl-CoA (ATP-citrate lyase) are the probably targets of the allatostatin, i.e., the first commit-ted steps in the synthesis of JH III.

2.9. Evolution of allatostatins

The molecular evolution of the allatostatin precursor in cockroaches has been analyzed recently using the gen-omic DNA sequences of the preproallatostatin precursor for four species of cockroach (Belles et al., 1990), as well as previously published sequences of two others (Donly et al., 1993; Ding et al., 1995). Phylogenetic analysis using parsimony revealed that the allatotropin sequences in these species were generated through a pro-cess of duplication of a single gene, similar to the situ-ation with the FMRFamides. This occurred before the

divergence of these species from each other during evol-ution and led to intragene families of peptides. A deter-mination of the physiological significance of such diver-sity is the next step.

2.10. Neurotransmitter effects on corpus allatum activity

If functional allatotropins/allatostatins cannot be identified in certain cases, such as the lack of Manduca allatotropin to stimulate larval glands, what else might regulate JH biosynthesis? Neurotransmitters are strong candidates, since the axons of non-neurosecretory neu-rons also innervate the corpora allata. There is evidence in both Manduca and Diploptera that neurotransmitters are regulatory factors, and this area of research deserves further attention. Octopamine, a primary insect neuro-transmitter, was shown a decade ago to stimulate JH biosynthesis in Locusta (Lafont-Cazal and Baehr, 1988) and later was found to have the same effect in honey bee adults (Kaatz et al., 1994) and larvae (Rachinsky, 1994). In Diploptera (Thompson et al., 1990) and the cricket Gryllus bimaculatus (Woodring and Hoffmann, 1994), JH production is inhibited by octopamine. Dopa-mine was subsequently found to affect JH synthesis in vitro by the corpora allata of adult female Blatella

germ-anica, and furthermore, its effect could be either

stimu-latory or inhibitory, depending on the stage of the female within the ovarian cycle (Pastor et al., 1991). At the pre-vious JH conference, dopamine was reported to have an effect on the corpora allata of Manduca sexta as well, and in a stage-specific manner during larval-pupal meta-morphosis (Granger et al., 1996). Early in the last larval stadium, dopamine stimulates both JH biosynthesis in vitro and cAMP production. After day 2, when a small ecdysteroid peak was observed prior to the commitment peak (Wolfgang and Riddiford, 1986), its effect on both JH and cAMP production was inhibitory. The source of dopamine affecting gland function has not yet been identified, but like the allatostatin, it has been found by immunocytochemistry in both the brain and corpora allata (Granger, unpublished information). When the corpora allata were screened for neurotransmitter content by electrochemical detection HPLC (Granger et al., 1996; T.C. Sparks, personal communication), the only biogenic amine detected was dopamine.

There is one recent line of research that implicates dopamine in the regulation of larval development, poss-ibly through an interaction with the corpora allata. Para-sitism of the armyworm, Pseudaletia separata, by the wasp Cotesia kariyai elevates levels of dopamine in the hemolymph and nerve cord, slows normal development, and delays pupation (Noguchi et al., 1995). These effects are caused by a biogenic peptide which is elevated in the hemolymph of the host (Noguchi and Hayakawa, 1996). Although the mechanism by which pupation is affected

has not yet been elucidated, persistent levels of JH are known to delay pupation in Lepidoptera. Thus, it is poss-ible that the elevated levels of dopamine may be affect-ing gland function. In the armyworm, Mamestra

bras-sicae, significantly higher levels of dopamine have been

found in the hemolymph and central nervous system of diapause-destined pupae (Noguchi and Hayakawa, 1997), and it would be of interest to know the primary site of dopamine action in this system as well.

2.11. Neurotransmitter receptors in the corpus allatum

Dopamine exerts its effects at the cell membrane via G protein-coupled receptors associated with cAMP (Kababian, 1992). The stage-specific effects of dopam-ine on JH biosynthesis by Manduca corpora allata are mirrored by dopamine effects on cAMP production (Granger et al. 1995b, 1996). This argues for (1) a direct effect of dopamine, rather than indirect (via the release of endogenous allatotropins/allatostatins), and (2) the existence of D1-like and D2-like dopamine receptors, the members of these families of receptors stimulating and inhibiting cAMP production. The results of a pharmaco-logical characterization of the dopamine receptors of the

Manduca corpora allata were presented at this meeting

(Granger et al., this symposium). On the basis of those results, it appears that the D2-like receptor(s) bears con-siderable similarity to those of vertebrates, while the D1-like receptor(s) is unusual in its limited recognition of a wide range of D1 and D2 receptor agonists and antagon-ists active in vertebrates. In this respect, the receptor is strikingly similar to a D1-like receptor isolated and sequenced in Drosophila (Gotzes et al., 1994; Sugamori et al., 1995).

2.12. Second messenger systems

In Diploptera, the effect of octopamine on corpora allata activity is paralleled by a concomitant effect on cAMP levels (Thompson et al., 1990), while diacylgly-cerol and 1,4,5-inositol triphosphate appear to be involved in transduction of the allatostatin signal (Rachinsky et al., 1994). Mas-AT stimulates inositol phosphate (IP) production in both male and female adult

Manduca corpora allata (Reagan et al., 1992), and it has

been proposed that the allatotropin stimulates JH pro-duction via an increase in 1,4,5-inositol triphosphate (IP3), leading to an increase in intracellular calcium.

High levels of intracellular calcium correlate with biosynthetically active glands in Manduca (Allen et al., 1992). Dopamine, by contrast, functions via cyclic AMP as its second messenger (Granger et al., 1996). The mechanism by which Mas-AS inhibits corpora allata activity in Manduca remains conjectural.

2.13. Interrelationship between neuropeptides and neurotransmitters in the control of the corpus allatum

The possible interrelationship between a neuro-transmitter and a neuropeptide in the control of JH biosynthesis is not a new observation, since it was shown several years ago that both octopamine and Dip-AS down-regulate JH biosynthesis in Diploptera (Thompson et al., 1990; Stay et al., 1994). Mechanisms proposed for the inhibition of JH synthesis by octopamine in

Diplop-tera include roles as (1) a neuromodulator of JH

syn-thesis, regulating ion channels or releasing allatostatin from terminals within the corpora allata; and (2) a neuro-hormone directly affecting JH biosynthesis. Thus, the relationship of dopamine to Mas-AS in inhibiting the corpora allata of Manduca larvae and adults is a critical consideration in the regulation of gland activity in this species.

2.14. Other factors affecting JH biosynthesis

Calcium is another factor impacting on the ability of the corpora allata to synthesize JH, and considerable work has been done in the last decade demonstrating the central and critical role of this ion in both Diploptera and Manduca (Rachinsky et al., 1994). There is a need for further information about the role of calcium in the transduction of neuropeptide and neurotransmitter sig-nals to the corpora allata, as well as of other intracellular events that would modulate the response.

One way to modulate the response of the glands to regulatory ligands is by controlling the kind and/or num-bers of their receptors. Ecdysteroid receptors are known to exist in the nuclei of Manduca corpora allata (Bidmond et al., 1992), and there is preliminary evidence that ecdysteroids control the switch in the responsiveness of the corpora allata to dopamine in Manduca, thus gov-erning the occurrence and/or numbers of D1- and D2-like receptors (Granger et al., 1996). Thus, the ecdys-teroids are strong candidates to figure in the overall scheme of the control of corpora allata function, but there are surprisingly few clues as to the mechanism by which this class of insect hormone is involved. With this decade’s accumulation of information about ecdysteroid receptors as well as transcription factors (Henrich et al., 1999), the stage is set for the investigation of the poss-ible control of corpora allata activity by ecdysteroids at the genomic level.

The ecdysteroid receptor is a heterodimeric complex of an ecdysteroid receptor protein (EcR) and ultraspira-cle protein (USP) (Yao et al., 1993; Thomas et al., 1993). While there have been no studies of the abundance of EcR-USP complexes or of their isoform diversity in the corpora allata, R. Rybczynski, P. Moshitsky, S. Apple-baum and L.I. Gilbert reported at this conference on the expression of EcR and USP gene products in the corpus

allatum of Manduca sexta, both in vitro and in vivo (unpublished information). Preliminary data indicate that the USP proteins in the glands are represented by a group of isoforms that probably represent the phos-phorylation states of two primary translation products. In the last larval stadium, the diversity of these isoforms is greatest at wandering, while the highest concentration of USP in the corpora allata occurs several days later, during the premolt peak of circulating ecdysteroids. EcR proteins were also present in multiple isoforms, again probably as the result of multiple phosphorylation states of primary translation products. In contrast to USP, how-ever, the EcRs in the corpora allata show few differences in relative abundance or isoform diversity during the fifth larval stadium. Various other larval tissues were also assayed and were found to be considerably different from the corpora allata in their USP and EcR isoform profiles, and some of these tissues expressed several putative EcR isoforms that varied with developmental stage. These results suggest that USP in the corpora allata and other tissues may form functionally diverse, tissue- and stage-specific homo- and/or heterodimers of the ecdysteroid receptor.

In vitro analysis indicated that USP isoform diversity in the corpora allata is altered in response to changes in physiological ecdysteroid levels, while ecdysteroid-dependent changes in EcRs in the glands are limited to changes in abundance. Very preliminary studies indicate that JH may also influence USP isoform diversity in the corpora allata, although the effect may not be direct. An understanding of the diversity of EcR and USP isoforms in the corpora allata will provide the basis for further study of the complex interactions between the ecydys-teroidogenic and JH biosynthetic pathways, as well as of the possible regulation of putative JH receptors and their downstream targets in the glands. Specific phos-phorylation events and the EcR and USP isoform diver-sity they generate are thus undoubtedly at the core of stage-specific responses of tissues and organs to the ecdysteroid titer. This provides an explanation for how a relatively small number of translation products and a limited number of ligands can be utilized effectively in a large number of permutations.

Thus the answer to the question originally posed by Carroll Williams and discussed at the First International Conference on the Juvenile Hormones in Lake Geneva, Wisconsin — Is JH the handmaiden of ecdysone? — may finally be within reach.

3. JH regulation: binding protein and metabolism

3.1. Nature of JHs

From the structure of JH III, it is obvious that there are a number of challenges that insects face in using this

compound as a hormone, i.e. the necessity to transport a highly lipophyllic molecule in an aqueous environment from its site of biosynthesis, through the insect hemo-lymph, to its site of action in target cells. JH biosynthesis and transport must therefore occur in what can be con-sidered a hostile environment, containing numerous esterases both outside and inside of the cell, capable of removing the conjugated methyl-ester of JH, and mem-brane bound epoxide hydrolases to degrade its stable C10,11 epoxide. Either of these metabolic events can significantly affect the pharmacology of JH and no doubt its biological action, discussed in more detail sub-sequently. Although these considerations of synthesis, transport and degradation are vital to JH function, the presence or absence of JH coordinated with changes in ecdysterioid levels at precise developmental time points is essential for normal growth, metamorphosis and reproduction. Insects have developed a unique system for the regulation of JH concentration which involves dynamic changes in two biochemical processes — JH biosynthesis and JH degradation. When JH biosynthesis levels are high, JH degradation is low, and when JH biosynthesis levels are low, JH catabolism is high. JH binding proteins play a critical role in these processes but at least in respect to the hemolymph, remain in molar excess in comparison to JH (Goodman and Gilbert, 1978). The challenge of JH regulation is further compli-cated by the synthesis of multiple JH homologs and by multiple sites of biosynthesis in some insect species. The biological significance of these observations is conjec-tural.

JH has been identified in approximately 100 insect species representing at least ten insect orders, with JH III being the predominant homolog (Baker, 1990). It would be useful in the future, and in terms of under-standing the role of JH in arthropod development in gen-eral, to examine what JH homologs if any are found in the most primitive insect orders, e.g. the Protura and Diplura. The higher homologs isoJH 0, JH I and JH II are found in the more advanced insect orders, i.e. the Lepidoptera, where in some cases multiple homologs are found in the same insect. In the cyclorrhaphous dip-terans, a new JH species, the bis-epoxide of JH III, was identified in Drosphila melanogaster, Calliphora

vom-itoria, Phormia regina and Lucilia cuprina (Richard et

al., 1989; Yin, 1994; Yin et al., 1995). There is no evi-dence that this JH III bis-epoxide is a degradation pro-duct of JH III (Moshitzky and Applebaum, 1995), and in fact, Yin et al. (1995) found that JH III, JH III bis-epoxide and methyl farnesoate had a synergistic effect on oogenesis in Phormia. Thus, the production of mul-tiple JH species has now been demonstrated in both the higher Diptera and the Lepidoptera. Methyl farnesoate is released from the corpora allata of a number of insect species (Cusson et al., 1991b). Sparagana et al. (1984) also showed that JH acid was produced by the corpora

world’s food supply. As Howard Schneiderman was

fond of saying “only a thin red line of entomologists

stands between humanity and disaster”.

How can one disregard the tremendous research

potential of Drosophila, whose entire genome has just

been sequenced, in addition to its many other

advan-tages. If only we could find a fly with a Manduca

epider-mis! It may be that the use of genetics (Drosophila) will

answer the general question of receptor-ligand

interac-tions, e.g. roles of other proteins comprising the

recep-tor-ligand complex that have escaped the vertebrate

endocrinologist working on mammals or chicks. In the

long term, we may be researching real uncharted

terri-tory rather than simulating and amusing our colleagues

who investigate the endocrinology of higher organisms.

Acknowledgements

We thank Pat Cabarga for excellent clerical assistance

and Dr. R. Rybczynski for the graphics of Figs. 3 and

4. The original research cited here from L. Gilbert’s

lab-oratory was supported by NIH grant DK-30018 and NSF

grant 9603710. The research of M. Roe’s laboratory was

supported by the USDA Competitive Grants Program,

NIH and the Herman Flasch Foundation.

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